The highly-lethal, pansystemic hemorrhagic fever caused by the mosquito-borne yellow fever virus (YFV) was one of the most feared diseases in Africa, Europe and the Americas, until the live-attenuated 17D vaccine was developed in the 1930's. The natural Asibi isolate of YFV was empirically passaged 176 times in primary cultured cells to derive the 17D virus. Although 17D is considered a prototypic live-attenuated virus vaccine, the molecular basis for its attenuation and immunogenicity remains unknown. In this proposal, recent developments in our understanding of flavivirus molecular biology and immunology will be exploited to identify and characterize determinants of YFV attenuation and expose molecular mechanisms that control the virus/host interaction. Genomic sequence comparisons revealed 48 nucleotide and 22 amino acid substitutions that occurred coincident with attenuation of 17D. in Aim 1, we will engineer chimeric viruses from cDNA clones of Asibi and 17D, and systematically map the attenuated phenotype of 17D: i) to structural or non-structural gene regions; ii) to specific genes; and finally iii) to single nucleotide/amino acid differences. Current models of arbovirus pathogenesis indicate that infection of dendritic cells (DCs) in the skin is a crucial early event, in which the viruses exploit migratory properties of activated DCs to effect viremic dissemination. Mice will be inoculated intradermally to mimic the natural route of infection (mosquito bite) or immunization, thereby providing an appropriate context to assess YFV virulence phenotype and pathogenesis. Since Asibi and 17D differ greatly in early viremic potential, we propose that differences in DC infection and activation are correlates of 17D attenuation. In Aims 2 and 3, we will test the hypothesis that controlled replication of 17D in DCs elicits an optimal immune response, whereas unrestricted Asibi replication dysregulates the inflammatory response with severe pathologic consequences. We will compare the permissivity of primary DC and macrophage cultures to infection with Asibi or 17D, and analyze global host gene regulation in response to virus infection. Finally, we will characterize the differential ability of Asibi and 17D viruses to target DCs in vivo, and compare the host's response to this infection in terms of DC activation and cytokine/chemokine induction profiles. These studies will begin to elucidate the molecular mechanisms of 17D attenuation and immunogenicity, and provide a framework for "rational" design of live-attenuated flavivirus vaccines.